Antibiotic stalobacins

ABSTRACT

Novel antibiotic stalobacins A to I having physico-chemical properties as shown in Tables 1 to 4 are provided, which are excellent antibiotics showing marked effects on Gram-positive bacteria.

This application is a continuation-in-part of now abandoned applicationSer. No. 08/111,045, filed Aug. 24, 1993.

The present invention relates to novel antibiotics. In particular, thisinvention relates to antibiotic stalobacins A, B, C, D, E, F and Gproduced by Pseudomonas sp. PBJ-5,360. In addition, this inventionrelates to antibiotic stalobacins H and I produced by Pseudomonas sp.PBJ-5360-STR-1-21. Furthermore, the invention relates to themicroorganisms which produce the antibiotics, and a process forproducing the same.

It is well known that antibacterial activity of a particular antibioticvaries depending on a nature of bacteria to be treated and the effect ofthe antibiotic often reduces because of the advent of resistant strains.The advent of multiple drug resistant bacteria has recently become a bigproblem. Accordingly, development of novel and effective antibiotics hasbeen desired for performing effective treatments. Above all, mayGram-positive bacteria, such as Staphylococcus, hemolytic streptococcusor the like are resistant to antibiotics, and there is continuous needfor the development of novel antibiotics having high potency to theseGram-positive bacteria.

It is disclosed in Japanese Patent Publication (Kokai) No. 303496/1990that Pseudomonas sp. PBJ-5,360 produces a complex of several antibioticswhich is therapeutically effective on Gram-positive bacteria. However,the publication does not disclose each of the components constitutingsaid complex. The inventors of the present invention have succeeded inisolating each of the components and determining physico-chemicalproperty of each component. The present invention is based on suchfindings.

Thus, the present invention provides antibiotic stalobacins selectedfrom the group consisting of stalobacins A, B, C, D, E, F and G producedby Pseudomonas sp. PBJ-5,360. These antibiotics are peptide antibioticswhich are produced by said Pseudomonas sp. PBJ-5,360. Stalobacins A-G(hereinafter sometimes referred to as merely "stalobacins") are producedin the form of a mixture of closely related analogs by cultivating saidstrain of Pseudomonas. Their antibacterial activities are much morepotent than those of known antibiotics. Stalobacins A-G havephysico-chemical properties as shown below in Tables 1-3.

The present invention further provides antibiotic stalobacins selectedfrom the group consisting of antibiotic stalobacins H and I produced bythe above-noted Pseudomonas sp. PBJ-5360-STR-1-21, a variant ofPseudomonas sp. PBJ-5360. These antibiotics are peptide antibioticswhich are produced by said Pseudomonas sp. PBJ-5360-STR-1-21.Stalobacins H and I (hereinafter sometimes also referred to as merely"stalobacins") are obtained in the form of a mixture of closely relatedanalogs by cultivating said Pseudomonas. Their antibacterial activitiesare much more potent than those of known antibiotics. Stalobacins H andI have physico-chemical properties as shown in the following Table 4.

                                      TABLE 1                                     __________________________________________________________________________    Physico-chemical Properties of Stalobacins A and B:                                            Stalobacin A                                                                             Stalobacin B                                      __________________________________________________________________________    m.p. (°C.) (as Na salt)                                                                 240° C. (dec.)                                                                    240° C. (dec.)                             LSI-MS Maximal Peak (m/z)                                                                      1483       1364                                              HRLSI-MS         1483.7347  1364.7165                                         (MH.sup.+) (m/z) C.sub.61 H.sub.107 N.sub.14 O.sub.28                                                     C.sub.58 H.sub.102 N.sub.13 O.sub.24              Theoretical Value                                                                              1483.7372  1364.7154                                         IR(KBr) (cm.sup.-1)                                                                            3385,1748  3389,1748                                                          1653,1526  1653,1526                                         UV (H.sub.2 O)   Terminal Absorption                                                                      Terminal Absorption                               (ε) at 210 nm                                                                          36,300     35,500                                            CD               [θ].sub.196 -77020                                                                 [θ].sub.193 -70720                                           [θ].sub.212 +3159                                                                  [θ].sub.209 +17630                                           [θ].sub.231 -33480                                                                 [θ].sub.231 -29070                                           [θ].sub.257 +4028                                                                  [θ].sub.257 +4905                           Retention Time (min.)                                                                          7.8        8.4                                               in HPLC*                                                                      Amino Acid Analysis (molar ratio)                                             HyAsp.sup.1)     HyAsp (1)  HyAsp (1)                                         Asp              Asp (1)    Asp (1)                                           Ser              Ser (2)    Ser (1)                                           HyIle.sup.2)     HyIle (1)  HyIle (1)                                         Gly              Gly (1)    Gly (1)                                           Ala              Ala (1)    Ala (1)                                           Arg              --         Arg (1)                                                            DNP - derivative                                                                         DNP - derivative                                                   m.p. >230° C. (dec.)                                                              m.p. >230° C. (dec.)                                        HRLSI-MS 1649.7378                                                                       HRLSI-MS 1530.7169                                                 C.sub.67 H.sub.109 N.sub.16 O32                                                          C.sub.64 H.sub.103 N.sub.15 O.sub.28                               Theoretical Value                                                                        Theoretical Value                                                  1649.7386  1530.7169                                         __________________________________________________________________________     *Column: Develosil 5C.sub.18, 4.6φ × 250 mm;?                       Mobile Phase: CH.sub.3 CN/2mM H.sub.3 PO.sub.4 (containing 50 mMNa.sub.2      SO.sub.4) = 43/57;?                                                           Flow Rate: 1 ml/min.;                                                         Chart Speed: 1 cm/min.                                                        .sup.1) Hydroxyaspartic acid                                                  .sup.2) Hydroxyisoleucine                                                

                                      TABLE 2                                     __________________________________________________________________________    Physico-chemical Properties of Stalobacins C and D:                                            Stalobacin C                                                                             Stalobacin D                                      __________________________________________________________________________    LSI-MS Maximal Peak (m/z)                                                                      1396       1309                                              HRLSI-MS         1396.7061  1309.6706                                         (MH.sup.+) (m/z) C.sub.58 H.sub.102 N.sub.13 O.sub.26                                                     C.sub.55 H.sub.97 N.sub.12 O.sub.24               Theoretical Value                                                                              1396.7053  1309.6732                                         IR(KBr)(cm.sup.-1)                                                                             3411,1744  3418,1745                                                          1652,1528  1646,1525                                         UV(H.sub.2 O)    Terminal Absorption                                                                      Terminal Absorption                               Retention Time (min.) in HPLC*                                                                 8.8        10.0                                              Amino Acid Analysis (molar ratio)                                             HyAsp.sup.1)     HyAsp (1)  HyAsp (1)                                         Asp              Asp (1)    Asp (1)                                           Ser              Ser (1)    --                                                HyIle.sup.2)     HyIle (1)  HyIle (1)                                         Gly              Gly (1)    Gly (1)                                           Ala              Ala (1)    Ala (1)                                           Arg              --         --                                                __________________________________________________________________________     *Column: Develosil 5C.sub.18, 4.6φ × 250 mm;                        Mobile Phase: CH.sub.3 CN/2mM H.sub.3 PO.sub.4 (containing 50 mMNa.sub.2      SO.sub.4) = 43/57;                                                            Flow Rate: 1 ml/min.;                                                         Chart Speed: 1 cm/min.                                                        .sup.1) Hydroxyaspartic acid                                                  .sup.2) Hydroxyisoleucine                                                

                                      TABLE 3                                     __________________________________________________________________________    Physico-chemical Properties of Stalobacins E, F and G                                           Stalobacin E                                                                          Stalobacin F                                                                          Stalobacin G                                __________________________________________________________________________    m.p. (°C.) (as Na salt)                                                                  240° C. dec.                                                                   240° C. dec.                                                                   240° C dec.                          LSI-MS Maximal Peak (m/z)                                                                       1378    1497    1485                                        HRLSI-MS          1378.7318                                                                             1497.7532                                                                             1485.7529                                   (MH.sup.+) (m/z)  C.sub.59 H.sub.104 N.sub.13 O.sub.24                                                  C.sub.62 H.sub.109 N.sub.14 O.sub.28                                                  C.sub.61 H.sub.109 N.sub.14 O.sub.28        Theoretical Value 1378.7311                                                                             1497.7529                                                                             1485.7529                                   IR(KBr)(cm.sup.-1)                                                                              3418,1748                                                                             3369,1746                                                                             3369,1746                                                     1651,1526                                                                             1653,1527                                                                             1654,1528                                   (UV) (H.sub.2 O)  Terminal                                                                              Terminal                                                                              Terminal                                                      Absorption                                                                            Absorption                                                                            Absorption                                  (ε) at 210 nm                                                                           39,500  36,480  37,500                                      CD                [θ].sub.194 -53530                                                              [θ].sub.196 -80050                                                              [θ].sub.196 -58600                                      [θ].sub.208 +15380                                                              [θ].sub.212 +1022                                                               [θ].sub.212 +10060                                      [θ].sub.231 -26750                                                              [θ].sub.231 -34490                                                              [θ].sub.231 -49830                                      [θ].sub.256 +4512                                                               [θ].sub.257 + 4788                                                              [θ].sub.257 +5621                     Retention Time (min.)*                                                                          12.4    11.7    17.0                                        in HPLC                                                                       Amino Acid Analysis (Molar Ratio)                                             HyAsp.sup.1)      HyAsp (1)                                                                             HyAsp (1)                                                                             HyAsp (1)                                   Asp               Asp (1) Asp (1) Asp (1)                                     Ser               Ser (1) Ser (2) Ser (2)                                     HyIle.sup.2)      HyIle (1)                                                                             HyIle (1)                                                                             HyIle (1)                                   Gly               Gly (1) Gly (1) Gly (1)                                     Ala               Ala (1) Ala (1) Ala (1)                                     Arg               Arg (1) --      --                                          __________________________________________________________________________     *Column: Develosil 5C.sub.18, 4.6φ × 250 mm;                        Mobile Phase: CH.sub.3 CN/2 mM H.sub.3 PO.sub.4 (containing 50 mMNa.sub.2     SO.sub.4) = 43/57;                                                            Flow Rate: 1 ml/min.;                                                         Chart Speed: 1 cm/min.                                                        .sup.1) Hydroxyaspartic acid                                                  .sup.2) Hydroxyisoleucine                                                

                  TABLE 4                                                         ______________________________________                                        Physico-chemical Properties of Stalobacins H and I:                                      Stalobacin H                                                                              Stalobacin I                                           ______________________________________                                        m.p. (°C.) (as Na salt)                                                             235° C. (dec.)                                                                       240° C. (dec.)                              LSI-MS Maximal Peak                                                                        1396          1325                                               (m/z)                                                                         IR (KBr) (cm.sup.-1)                                                                       3374, 1747,   3387, 1747,                                                     1654, 1597, 1525                                                                            1651, 1596, 1527                                   UV (H.sub.2 O)                                                                             Terminal absorption                                                                         Terminal absorption                                CD (H.sub.2 O)                                                                             [θ].sub.194 -66980                                                                    [θ].sub.206 +11530                                        [θ].sub.212 +9851                                                                     [θ].sub.232 -28660                                        [θ].sub.232 -31520                                                                    [θ].sub.257 +4749                                         [θ].sub.257 +4288                                          Retention time (min.)                                                                      8.8           9.7                                                in HPLC*                                                                      Amino Acid Analysis                                                           (molar ratio)                                                                 HyAsp.sup.1) HyAsp (1)     HyAsp (1)                                          Asp          Asp (1)       Asp (1)                                            Ser          Ser (1)       Ser (1)                                            HyIle.sup.2) HyIle (1)     HyIle (1)                                          Gly          Gly (1)       Gly (1)                                            Ala          Ala (1)       --                                                 ______________________________________                                         *Column: Develosil 5C18, 4.6 i.d. × 250 mm                              Mobile phase: CH.sub.3 CN/2 mM H.sub.3 PO.sub.4 (containing 50 mM Na.sub.     SO.sub.4) =  43/57                                                            Flow rate: 1 ml/min.                                                          .sup.1) Hydroxyaspartic acid                                                  .sup.2) Hydroxyisoleucine                                                

The antibiotic stalobacins of the present invention characterized by theabove properties have been found to have excellent antibacterialactivities in vitro and in vivo, showing potent effects especially onGram-positive bacteria.

Thus, the antibiotic stalobacins of the present invention show excellentactivities, and have higher activities especially against Gram-positivebacteria, as shown below in Tables 7 and 9. Regarding stalobacins A-G,the mechanism is considered to be based on the inhibition of cell wallsynthesis.

No death was observed in acute toxity test by intravenous administrationof 300 mg/kg and 500 mg/kg of the antibiotic stalobacins to mice.

The antibiotic stalobacins A-G of the present invention are produced bycultivating Pseudomonas sp. PBJ-5,360 belonging to Genus Pseudomonasaerobically in liquid medium containing assimilable carbon sources,nitrogen sources and mineral salts in a conventional manner. Thisbacterium has been identified as the above-mentioned strain bycultivating it according to the method as hereinafter described inExperiment 2 and examining comprehensively its morphology, cultureproperties, physiological and biochemical properties in reference to thedescription of Bergy's Manual of Systematic Bacteriology, Vol. 1 (1984).This strain may undergo a spontaneous or artificial mutation, and it isobvious to a person skilled in the art that such mutants should beincluded in the scope of the present invention, as far as they retain anability to produce the stalobacins of the present invention. Thus, thepresent invention provides also Pseudomonas sp. PBJ-5,360 producingnovel antibiotic stalobacins and mutants thereof having an ability toproduce said antibiotic stalobacins. Pseudomonas sp. PBJ-5,360 wasdeposited under accession No. FERM P-10578 with National Institute ofBioscience and Human Technology, Higashi 1-1-3, Tsukuba-shi, IbarakiPref. Japan, on Feb. 27, 1989, and then transferred to InternationalDeposition under Budapest Treaty on Jun. 17, 1993, and assignedaccession No. FERM BP-4342.

Antibiotic stalobacins H and I of the present invention are produced bycultivating Pseudomonas sp. PBJ-5360-STR-1-21, a variant derived fromPseudomonas sp. PBJ-5360 (BIKOKEN deposition No. 10578, FERM BP-4342),which produces a mixture of stalobacins, aerobically in a liquid mediumcontaining assimilable carbon sources, nitrogen sources and mineralsalts in a conventional manner. This bacterium has been identified asthe above mentioned strain by cultivating it according to the method ashereinafter described in Experiment 4 and examining comprehensively itsmorphology, culture properties, physiological and biochemical propertiesin reference to the description of Bergey's Manual of SystematicBacteriology, Vol. 1 (1984). This strain may undergo a spontaneous orartificial mutation, and it is obvious to a person skilled in the artthat such mutants should be included in the scope of the presentinvention, as far as they retain an ability to produce the stalobacinsof the present invention. Thus, the present invention provides alsoPseudomonas sp. PBJ-5360-STR-1-21 producing novel antibiotic stalobacinsH or I and mutants thereof having an ability to produce said antibioticstalobacins. Pseudomonas sp. PBJ-5360-STR-1-21 was deposited underaccession No. FERM BP-4661 with National Institute of Bioscience andHuman Technology, Higashi 1-1-3, Tsukubashi, Ibaraki Pref. Japan, onApr. 28, 1994.

Furthermore, the present invention provides a process for producingantibiotic stalobacins A to I by cultivating such strains.

Ordinary compositions of medium and ordinary conditions used forconventional cultivation for producing antibiotics can be adopted. Inprinciple, the medium includes carbon sources, nitrogen sources, mineralsalts and the like. If necessary, vitamins, precursors or the like canbe added. Examples of carbon sources are glucose, starch, dextrin,glycerin, molasses, organic acids and the like, and these carbon sourcesmay be used alone or in a mixture thereof. Examples of nitrogen sourcesare soybean powder, corn steep liquor, meat extract, yeast extract,cotton seed powder, peptone, wheat embryos, ammonium sulfate, ammoniumnitrate and the like, and these nitrogen sources may be used alone or ina mixture thereof. Examples of mineral salts are calcium carbonate,sodium chloride, potassium chloride, magnesium sulfate, cupric sulfate,manganese chloride, zinc sulfate, cobalt chloride, various phosphatesand the like. These mineral salts may be added to a medium whenrequired. A sufficient amount of antibiotic stalobacins A to G isproduced by cultivating Pseudomonas sp. PBJ-5,360 of the presentinvention in an appropriate medium at temperature from 20° to 35° C.,preferably 25°-29° C., for about 1-7 days. Similarly, a sufficientamount of antibiotic stalobacins H and I is produced by cultivatingPseudomonas sp. PBJ-5360-STR-1-21 of the present invention in anappropriate medium at temperatures from 20° to 35° C., preferably 25° to29° C., for about 1 to 7 days. The product is then isolated andpurified, if necessary, from the culture in a conventional manner. Allof such procedures are well known to a person skilled in the art.

The antibiotic stalobacins of the present invention are believed to beuseful for treating various infections, in particular, treatinginfections caused by multiple drug-resistant Gram-positive bacteria,since they exhibit marked antibacterial activities in vivo and in vitro.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows IR spectrum of stalobacin A.

FIG. 2 shows IR spectrum of stalobacin B.

FIG. 3 shows IR spectrum of stalobacin C.

FIG. 4 shows IR spectrum of stalobacin D.

FIG. 5 shows IR spectrum of stalobacin E.

FIG. 6 shows IR spectrum of stalobacin F.

FIG. 7 shows IR spectrum of stalobacin G.

FIG. 8 shows NMR spectrum of DNP-stalobacin A.

FIG. 9 shows NMR spectrum of DNP-stalobacin B.

FIG. 10 shows NMR spectrum of stalobacin E.

FIG. 11 shows NMR spectrum of stalobacin F.

FIG. 12 shows NMR spectrum of stalobacin G.

FIG. 13 shows high performance liquid chromatogram demonstrating mutualconversion between stalobacins A and A' and stalobacins B and B'.

FIG. 14 is a graph showing IR spectrum of stalobacin H.

FIG. 15 is a graph showing IR spectrum of stalobacin I.

FIG. 16 is a graph showing NMR spectrum of stalobacin H.

FIG. 17 is a graph showing NMR spectrum of stalobacin I.

The present invention will be explained in more detail below byillustrating Examples and Experiments.

EXAMPLE 1

(a) Fermentation Step:

Eight hundreds ml of a medium (adjusted to pH 7 with 2N-NaOH) consistingof 1.0% glucose, 0.5% yeast extract (Difco) and tap water in a 2 LErlenmeyer flask was inoculated with Pseudomonas sp. PBJ-5,360 (kept at-80° C. in 2 ml vial), and the resultant mixture was subjected to ashaking cultivation at 180 rpm with 70 mm of shaking breadth attemperature of 28° C. for 20 hours. The culture (800 ml) was implantedto 25 L of a medium (adjusted to pH 7 with 2N-NaOH) containing 1.0%glucose, 0.4% yeast extract (Difco), 1.0% molt extract (Difco), 0.1%polypeptone (Nippon Seiyaku) and tap water in 50 L jar fermenter, andthe resultant mixture was cultivated with agitation at 200 rpm, with 20L/min of aeration, under 0.35 kg/cm² of inner pressure, at temperatureof 28° C. for 20 hours.

Then, 13 L of this culture was implanted to 250 L of a medium (adjustedto pH 7 with 2N-NaOH) consisting of 1.0% glucose, 1.0% molt extract(Difco), 1.0% powdery yeast, 0.2% tomato paste (Kagome), 1.0%β-cyclodextrin, 0.0008% defoaming agent P-2000 (Dainippon Ink) and tapwater in a 500 L tank, and the resultant mixture was cultivated with 120L/min of aeration, under 0.35 kg/cm² of inner pressure, with agitationat 320 rpm at a temperature of 28° C. for 94 hours.

(b) Separation Step:

To 375 L of the culture obtained in the foregoing step was added 3.7 Lof chloroform for sterilization. Then, the mixture was adjusted to pH8.5 with 2N-NaOH, mixed with 27 L of Diaion HP-20 (Mitsubishi KaseiCorporation) and stirred for 4 hours for adsorbing the active substancesonto the resin. The mixture was allowed to stand for 1 hour, and thesupernatant was discarded to recover Diaion HP-20. The resin was washedwith water and put in a glass column (inner diameter: 30 cm), and theactive substances were eluted by a gradient of 40 L of 10 mM Na₂ HPO₄-40 L of 90% methanol.

The fraction (21 L) showing antibacterial activity to S. aureus JC-1 wascollected, adjusted to pH 7.0 with 2N-HCl and concentrated in vacuo to 3L. The concentrate was washed with 3 L of ethyl acetate to removelipophilic materials. The ethyl acetate contained in the aqueous layerwas evaporated in vacuo and the residue was charged onto MCI-GEL CHP 20P(Mitsubishi Kasei Corporation) column (5 cm I.D.×51 cm) to adsorb theactive substances, and the active substances were eluted stepwise with 2mM Na₂ HPO₄ -80% MeOH.2 mM Na₂ HPO₄. The eluted fractions were subjectedto a HPLC analysis, and the fractions containing a group of stalobacinswere collected, adjusted to pH 7.0 with 2N-HCl, concentrated in vacuoand lyophilized. 3490 mg.

(c) Purification Step:

The First Purification Step:

Two lots of the crude extract obtained in the foregoing step werecombined and dissolved in 230 ml of water (pH 8). The solution wasadsorbed on Whatman DE-52 (Cl) ion exchange cellulose column (2 cmI.D.×50 cm), and the column was washed with water. The column wassubjected to a gradient elution with 600 ml of 10 mM Na₂ HPO₄ (pH7.5)-600 ml of 1 M NaCl.10 mM Na₂ HPO₄ (pH 7.5). The eluted fractionswere subjected to a HPLC analysis, and 370 ml of the fractionscontaining a group of stalobacins were collected and adjusted to pH 7.0with 2N-HCl. The above fraction (370 ml) was adsorbed on MCI-GEL CHP 20P(Mitsubish Kasei Corporation) column (2 cm I.D.×50 cm), which was thensubjected to a gradient elution with 10 mM Na₂ HPO₄ (pH 7.5)-90% MeOH.10mM Na₂ HPO₄ (pH 7.5). The eluted fractions were subjected to a HPLCanalysis, and the fractions containing a group of stalobacins werecollected, adjusted to pH 7.0 with 2N-HCl, concentrated in vacuo andlyophilized.

                  TABLE 5                                                         ______________________________________                                        Fraction *No.                                                                            mg         Ingredient                                              ______________________________________                                         1-63      Ca. 0                                                              63-75      1400       Stalobacins B, E                                        76-90       600       Stalobacins A, B                                         91-134    1250       Stalobacins A, C, D, F, G                               ______________________________________                                         *15 ml/Fraction                                                          

The Second Purification Step:

The fractions obtained above were purified by the preparative highperformance liquid chromatography under the following conditions andthen by the recycle preparative high performance liquid chromatographyto give stalobacins A, B, C, D, E, F and G.

Fractions 63-75 (1400 mg) were dissolved in 40 ml of pure water, and thesolution was subjected to the preparative high performance liquidchromatography with YMC ODS column (s-15/30μ, 5.0×5.0 cm, eluent:acetonitrile-2 mM phosphoric acid (50 mM Na₂ SO₄) 47:53, flow rate: 100ml/min, detection: 220 nm) to give a fraction (1.2 L) containingstalobacin B as a main ingredient and a fraction (2.0 L) containingstalobacin E as a main ingredient. Each fraction was neutralized withdilute aqueous sodium hydroxide and the acetonitrile was evaporated. Theresidue was adjusted to pH 7.4-7.5 and adsorbed on a column of MIC-GELCHP-20P, which was washed with water and eluted with 60% aqueousacetone. Eluates were concentrated in vacuo and lyophilized to give 92mg of stalobacin B fraction and 20 mg of stalobacin E fraction. Thestalobacin B fraction (92 mg) obtained above was subjected to therecycle preparative high performance liquid chromatography withDevelosil 5 C₁₈ column (2.0×25 cm, eluent: acetonitrile-2 mM phosphoricacid (50 mM Na₂ SO₄) 47:53, flow rate: 10 ml/min., detection: 220 nm)(twice repeated). The fraction separated was concentrated to remove theacetonitrile, and the residue was neutralized with dilute sodiumhydroxide, adjusted to pH 7.3-7.5, adsorbed on a column of MIC-GELCHP-20P, which was then washed with water and eluted with 60% aqueousacetone. The eluate was concentrated in vacuo to remove the acetone, andthe residue was lyophilized to give 42 mg of pure stalobacin B. Further,the stalobacin E fraction (20 mg) obtained above was subjected to therecycle preparative high performance liquid chromatography (twicerepeated) using Develosil 5 C₁₈ column in the same manner as describedabove to give 9 mg of pure stalobacin E.

Fractions 91-134 (1250 mg) obtained in the first purification step weretreated in the same manner as above to give pure stalobacins A (83 mg),C (2 mg), D (3 mg), F (19 mg) and G (12 mg).

EXAMPLE 2.

Preparation of DNP-stalobacins A and B: A mixture of stalobacins A and B(203 mg) was dissolved in 20 ml of 50% aqueous methanol, mixed with 800mg of sodium bicarbonate and 4 ml of 10% ethanolic solution of2,4-dinitrofluorobenzene and stirred at room temperature for 2 hours.The reaction mixture was adjusted to pH 3.0 with dilute hydrochloricacid, and the aqueous layer was washed with ether and ethyl acetatesuccessively and extracted with n-butanol. The organic layer was driedover sodium sulfate and concentrated in vacuo. The residue (240 mg) wassubjected to high performance liquid chromatography to give 65 mg ofDNP-stalobacin A (reaction product of 2,4-dinitrofluorobenzene andstalobacin A)(t_(R) 18 min.) and 29 mg of DNP-stalobacin B (t_(R) 20.2min.).

Separation condition:

Column=Develosil ODS 2.0×25 cm

Eluent=44% acetonitrile-2 mM phosphate buffer (pH 7.5, containing 50 mMNa₂ SO₄)

DNP-stalobacin A: m.p. >230° C.

HR-LSIMS (MH⁺) 1649.7378

C₆₇ H₁₀₉ N₁₆ O₃₂ Theoretical Value 1649.7386

DNP-stalobacin B: m.p. >230° C.

HR-LSIMS (MH⁺) 1530.7169

C₆₄ H₁₀₄ N₁₅ O₂₈ Theoretical Value 1530.7169

Physico-chemical properties of stalobacins A, B, C, D, E, F, G,DNP-stalobacin A and DNP-stalobacin B obtained in Examples 1-2 were asshown in Tables 1, 2 and 3. IR spectra of stalobacins A-G were shown inFIGS. 1-7, and NMR spectra of DNP-stalobacin A, DNP-stalobacin B,stalobacin E, stalobacin F and stalobacin G were shown in FIGS. 8-12 ofthe accomanying drawings, respectively. Putative partial structures ofDNP-stalobacin A and DNP-stalobacin B were shown below. ##STR1##

In the chemical structures above, HyAsp and HyIle representhydroxyaspartic acid and hydroxyisoleucine, respectively, and (HyAsp)represents a moiety including hydroxyaspartic acid, and X¹, X², X³, X⁴and X⁵ represent unidentified moieties. There is possibility that all orsome of X¹, X², X³, X⁴ and X⁵ represent a single bond and they connectto the C₁₄₋₁₅ H₂₄₋₂₅ N₄ O₁₃ (HyAsp) moiety in the molecule.

Unique Properties of Stalobacins A and B:

(1) As shown in FIG. 13(a), a mixture of stalobacins A and B wassubjected to high performance liquid chromatography, whereby total 4peaks corresponding to A, its isomer A', B and its isomer B' wereobserved However, when this mixture was allowed to stand for 24 hoursand then subjected again to high performance liquid chromatography, onlytwo peaks corresponding to A and B were observed with disappearance ofpeaks of A' and B'. Each of A and B was isolated and again subjected toliquid chromatography, whereby two peaks corresponding to A and A' wereobserved for A and two peaks corresponding B and B' were observed for B(FIG. 13(b)). Each of these fractions was allowed to stand for one day,whereby the peaks of A' and B' disappeared again (FIG. 13(c)).

(2) Stalobacin B has a molecular formula C₅₈ H₁₀₁ N₁₃ O₂₄ and an IRabsorption at 1747 cm⁻¹. It is treated with dilute NaOH to give acompound of molecular formula C₅₈ H₁₀₃ N₁₃ O₂₅. The antibacterialactivity markedly lowered with the disappearance of the absorption at1747 cm⁻¹.

(3) Table 6 shows the aspect of the mutual conversion between A and A'when stalobacin A was dissolved in aqueous solution of various pH.

                  TABLE 6                                                         ______________________________________                                               Composition (%)*                                                       Time after                                                                             pH3.0       pH7.0       pH8.0                                        dissolution                                                                            A       A'      A     A'    A     A'                                 ______________________________________                                        0        82.2    17.8    81.9  18.1  82.1  17.9                               1 hour   96.0    3.1     87.9  12.1  87.3  12.7                               2 hour   100     0       100   0     100   0                                  3 hour   100     0       100   0     100   0                                  ______________________________________                                         *High Performance Liquid Chromatography                                  

EXAMPLE 3

(a) Fermentation Step:

Eight hundreds ml of a medium (adjusted to pH 7 with 2N-NaOH) consistingof 1.0% glucose, 0.5% yeast extract (Difco) and tap water in a 2 LErlenmeyer flask was inoculated with a seed strain of Pseudomonas sp.PBJ-5360-STR-1-21 (kept at -80° C. in a 2 ml vial), and the resultantmixture was subjected to a shaking cultivation at 180 rpm with 70 mm ofshaking breadth at 28° C. for 22 hours. The culture (800 ml) wasimplanted to 20 L of a medium (adjusted to pH 7 with 2N-NaOH) containing1.0% glucose, 0.4% yeast extract (Difco), 1.0% malt extract (Difco),0.1% polypeptone (Nippon Seiyaku) and tap water in a 30 L jar fermenter,and the resultant mixture was cultivated with agitation at 200 rpm, with14 L/min of aeration under 0.35 kg/cm² of inner pressure, at 28° C. for21 hours.

Then, 8 L of this culture was implanted to 125 L of a medium (adjustedto pH 7 with 2N-NaOH) consisting of 2.0% soluble starch, 2.0% powderyyeast, 1.5% β-cyclodextrin, 0.5% olive oil, 0.3% magnesium chloride.6H₂O, 0.1% potassium primary phosphate, 0.0008% defoaming agent ADECANOL(Asahi Denka Kogyo) LG109 and tap water in a 250 L tank, and theresultant mixture was cultivated with 65 L/min of aeration, under 0.35kg/cm² of inner pressure, with agitation at 350 rpm at 28° C. for 72hours.

(b) Separation Step:

To 138 L of the culture obtained in the foregoing step was added 1.4 Lof chloroform for sterilization. Then, 15 L of Amberlite XAD-7 (Organo)was added and the resulting mixture was mixed with stirring for 3 hoursfor a batch adsorption of the active substances onto the resin. Theresin was recovered using a 0.1 mm mesh stainless steel filter. Theresin was washed with water, put in a glass column (inner diameter: 20cm), washed with 40 L of water, 40 L of 30% methanol and then 15 L of50% methanol in 20 mM phosphate buffer (pH 7.5), and the activesubstances were eluted by 40 L of 60% methanol in 20 mM phosphate buffer(pH 7.5).

Fractions (20 L) showing antibacterial activity to S. aureus JC-1 werecollected, concentrated in vacuo to 3 L. The concentrate was washed with3 L of ethyl acetate to remove lipophilic materials. The ethyl acetatecontained in the aqueous layer was evaporated in vacuo. The activesubstances were adsorpted on Amberlite XAD-7 (Organo) in a 1 L column(inner diameter 6.5 cm×31 cm) and the resin was washed with 2 L ofwater. The elution was carried out using 2 L of 30% aqueous MeOH and 3 Lof 50% aqueous MeOH. The eluted fractions were subjected to a HPLCanalysis, and the fractions containing stalobacins were collected,adjusted to pH 7.0 with 2N-HCl, concentrated in vacuo and lyophilized toobtain 3890 mg of powder.

(c) Purification Step:

The First Purification Step:

One thousand and eight hundred twenty mg of the crude powder obtained inthe foregoing step was dissolved in 60 ml of 20 mM phosphate buffer (pH7.5). The solution was subjected to a preparative high-speed liquidchromatography using YMC ODS column [S-15/30μ, 5.0×50 cm, eluent:acetonitrile/20 mM phosphate buffer (pH 7.5). 50 mM sodiumsulfate=40/60, flow rate: 50 ml/min, UV detection; 210 nm] to obtain afraction (2.4 L) containing stalobacins H and I as main ingredients. Theacetonitrile in the fraction was distilled off and the residue waspassed through a column of Diaion HP-20 (Mitsubishi Kasei Corporation).The resin was washed with water, and the adsorbed components were elutedwith 60% aqueous acetone. The acetone in the eluate was distilled awayin vacuo and the residue was lyophilized to obtain 126 mg of powder.

The Second Purification Step:

The powder obtained in the above step was purified by preparativehigh-speed liquid chromatography under the following conditions toobtain stalobacins H and I.

The powder (126 mg) was dissolved in 7 ml of 50 mM phosphate buffer (pH7.0). Fifteen mg of the sample per one procedure was charged intoAsahipak ODP-90 column (inner diameter 21.5 mm×300 mm, eluent:acetonitrile solution of 20 mM AcOH/aqueous solution of 20 mMAcOH=40/60, flow rate: 8 ml/min, UV detection: 220 nm), and stalobacin Hwas collected from the fractions of 144 ml to 184 ml and stalobacin Iwas collected from the fractions of 216 ml to 288 ml. This fractionationwas repeated, and collected fractions were neutralized to pH 7.0 withaqueous 1N-NaOH. The acetonitrile was distilled off in vacuo and NaClwas added to the residue to obtain 5% NaCl concentration. The resultantmixture was adjusted to pH 7.5 with 1N NaOH and passed through MCI GELCHP20P column (75 to 150μ, Mitsubishi Kasei) which had been equilibratedwith 5% aqueous NaCl solution. The column was washed with water andeluted with 70% aqueous MeOH. The MeOH in the eluate was distilled offin vacuo and the residue was lyophilized to give 12 mg of purestalobacin H and 38 mg of pure stalobacin I.

Physico-chemical properties of stalobacins H and I obtained in Example 3are shown in Table 9. IR spectra of stalobacins H and I were shown inFIGS. 14 and 15 respectively, and NMR spectra of stalobacins H and Iwere shown in FIGS. 16 and 17 respectively.

Experiment 1 Antibacterial Activity in vitro and in vivo:

1) In vitro Antibacterial Activity:

Antibacterial activity in vitro of antibiotic stalobacins A-G obtainedin Example 1 was assayed by the agar dilution method. Table 7 shows theresults.

                  TABLE 7                                                         ______________________________________                                        Gram-positive                                                                              Stalobacin (μg/ml, 10.sup.6 cfu/ml)                           bacteria     A      B      C    D    E    F    G                              ______________________________________                                        S. aureus FDA JC-1                                                                         0.025  0.1    0.08 0.01 0.025                                                                              0.02 0.1                            S. faecalis SR1004                                                                         0.1     0.39  0.31 0.08 0.39 0.1  0.2                            S. aureus 3626                                                                             0.05   0.1    0.16 0.04 0.025                                                                              0.05 0.2                            (MRSA)                                                                        ______________________________________                                    

2) In vivo Antibacterial Activity:

Antibacterial activity in vivo of stalobacin A was assayed. Mice wereintraperitoneally challenged with infectious bacteria. One hour afterthe challenge, the test compound was subcutaneously administered. ED₅₀value was calculated on the basis of survival ratio on 7th day after thechallenge. MIC was determined according to the agar dilution method.Table 8 shows the results.

                  TABLE 8                                                         ______________________________________                                        Protective Effect of Stalobacin A in Mice Systemically Infected:                             ED.sub.50 (mg/kg)                                                                       MIC (μg/ml)                                                      Stalobacin A                                                                            Stalobacin A                                         ______________________________________                                        S. aureus Smith  0.17        0.006                                            S. aureus SR3637 (H-MRSA)                                                                      0.27        0.025                                            S. pyogenes C-203                                                                              0.12        0.013                                            S. pneumoniae Type I                                                                            0.058      0.006                                            ______________________________________                                    

Experiment 2 Bacteriological Properties of PBJ-5,360:

Bacteriological properties of PBJ-5,360 of the present invention areshown below. PBJ-5,360 was isolated from the soil collected in Kyoto,Japan.

Cultivation was effected at 28° C. in principle.

A. Morphology:

It is a Gram-negative rod. Its size is 0.3-0.5 (μ)×0.8-1.3 (μ). Itvigorously moves with one or more polar flagella.

B. Characteristics of Culture

1) Cultivation in meat infusion medium:

Growth of the bacteria was hardly observed. Off white translucentprecipitates formed very slightly at the bottom of the test tube.

2) Meat infusion agar stab culture:

Growth in thread form or small nipple form along the stab line wasobserved. Neither evolution of gas nor production of pigment wasobserved. Reddish thin bacterial plaque appeared on the surface, butthis bacterial plaque became translucent and light brown with the lapseof time and verrucose projections were observed in several places. It isan aerobic bacterium.

3) Meat infusion agar slant culture:

The growth of the bacteria was not so rapid and began at 28° C. aftertwo days (observed with naked eyes). The bacteria grew in thread form,and the bacterial plaque was translucent and light yellow with flatswelling having a spotty appearance. The periphery was whole peripheral.Then, the bacterial plaque grew favorably in thread form or verrucoseform with gloss. Thus, a wet slightly reddish translucent brownbacterial plaque was obtained. The periphery was wavy or long wavy.Production of any gas or pigment was not observed.

4) Meat infusion gelatin stab culture.

Cultivation was effected at room temperature (22°-25° C.). The gelatinwas liquefied.

5) Cultivation on the meat infusion agar plane medium:

The growth of the bacteria was not so rapid. The colony became visibleat 28° C. after two days. The colony was initially small, spotty,translucent and brown with a whole periphery. The colony was too smallto be observed about its swelling. Then, the colony grew in a spotty orcircular form and with whole periphery and the swelling was flat orconvex circular. The colony was translucent and brown with gloss.Neither gas nor soluble pigment was produced.

6) Characteristics in litmus milk culture:

An acid formation and peptonization occurred, but the reactions occurredrather slowly. Thus, the reaction began after 14 days. No gas evolved.Sometimes grey reddish purple thin pellicle formed on the surface. Theupper layer was translucent and reddish purple, and the lower layer wasopaque and purplish beige. The precipitate was reddish purple close tobeige.

C. Physiological and Biochemical Properties:

1) Catalase test: positive

2) Oxidase test: positive

3) OF-test: negative (showing to be alkaline)

4) Hemolytic test: positive (weakly)

5) Viability at 5° C.: negative

6) Production of H₂ S: negative

7) Reducing ability for nirate: positive

8) Denitrification: negative (although no nitrogen gas was evolved, itseemed to reduce NO₂.)

9) Availability of citric acid: negative (Christensen medium and Simonsmedium)

10) Growth on NAC agar medium: negative (nonviable)

11) Production of indole: negative

12) Voges-Proskauer reaction (Voges-Proskauer test): negative

13) Methylred test: negative

14) Hydrolyzing ability for arginine: positive

15) Decarboxylation ability for lysine: positive

16) Decarboxylation ability for ornithine: positive

17) Hydrolyzing ability for esculine: negative

18) DNase test: negative

19) Hydrolyzing ability for starch: negative

20) ONPG test (cultivated at 37° C.): negative

21) Acylamidase test: negative

22) Phosphatase test: positive

23) Hydrolyzing ability for chitin: negative

24) Productivity of levan from sucrose: positive

25) Productivity of acids and gas from sugars:

Neither acids nor gases were produced from the following 13 sugars:glucose, fructose, galactose, mannose, xylose, arabinose, maltose,lactose, ramnose, sucrose, cellobiose, trehalose, mannit.

26) Accumulation of poly-β-hydroxybutyrate in the cell: negative

27) Availability of carbon sources: On the medium containing minerals,glucose and calcium 2-keto-gluconate can be used as a sole carbon sourcefor the formation of the cells. In this case, it seemed that specificvitamines for growth were not required. On the other hand,D-(+)-trehalose, DL-arginine, geraniol, β-alanine, L-valine and innositcan not be used.

28) G+C mole % (HPLC method): 60.4% (A+T mole %=39.6%)

In view of the properties above, the present bacterium is an aerobicGram-negative rod and moves actively in a liquid medium using one ormore polar flagella. It is positive for catalase and contains oxidase.It was negative for OF-test (showing to be alkaline). In view of theseobservations, it is apparent that the present bacterium belongs to GenusPseudomonas in Family Pseudomonadaceae.

When the inventors compared the above properties with those of thebacterial complexes which are incapable of accumulatingpoly-β-hydroxybutyrate (PHB) in their cells, which complexes aredescribed in Bergey's Manual of Systematic Bacteriology, Vol. 1 (1984)on Genus Pseudomonas, the present inventors failed to find any bacteriumhaving those properties consistent or analogous to the propertiesdescribed above. Therefore, this bacterium may be a new strain ofPseudomonas, and it appears a considerably unusual strain of Pseudomonasbecause it hydrolyzes arginine and decarboxylates lysine and ornithine.The G+C mole % value of 60.4% indicates that the strain belongs to agroup having lower G+C value in Pseudomonas. Thus, in view of thevarious properties above, the present bacterium has been designated asPseudomonas sp. PBJ-5,360.

Experiment 3 Antibacterial Activity in vitro and in vivo

1) In vitro Antibacterial Activity

Antibacterial activity in vitro of antibiotic stalobacins H and Iobtained in Example 3 was assayed by the agar dilution method. Theresults are shown in Table 9.

                  TABLE 9                                                         ______________________________________                                                     Stalobacin (μg/ml), 10.sup.6 cfu/ml)                          Gram-positive bacteria                                                                       H           I                                                  ______________________________________                                        S. aureus FDA JC-1                                                                           0.1          0.05                                              S. faecalis SR1004                                                                           0.2         0.2                                                S. aureus 3626 (MRSA)                                                                        0.1         0.1                                                ______________________________________                                    

2) In vivo Antibacterial Activity

Antibacterial activity in vivo of stalobacin I was assayed. Mice wereintraperitoneally challenged with infectious bacteria. One hour afterthe challenge, the test compound was subcutaneously administered. ED₅₀values was calculated on the basis of survival ratio on 7th day afterthe challenge. MIC was determined according to the agar dilution method.The results are shown in Table 10.

                  TABLE 10                                                        ______________________________________                                        Protective Effect of Stalobacin I in Mice Systemically                        Infected:                                                                                    ED.sub.50 (mg/kg)                                                                       MIC (μg/ml)                                                      Stalobacin I                                                                            Stalobacin I                                         ______________________________________                                        S. aureus SR3637 (H-MRSA)                                                                      0.12        0.012                                            S. pneumoniae Type I                                                                           0.046       0.006                                            E. faecalis SR1004                                                                             1.50        0.2                                              ______________________________________                                    

Experiment 4 Bacteriological Properties of PBJ-5360 andPBJ-5360-STR-1-21:

PBJ-5360-STR-1-21 of the present invention was obtained as a mutant ofthe above-mentioned PBJ-5360 strain. PBJ-5360 was isolated from the soilcollected in Kyoto, Japan. Various bacteriological properties ofPBJ-5360-STR-1-21 of the present invention are shown below. Cultivationwas effected at 28° C. in principle.

A. Morphology:

It is a Gram-negative rod. Its size is 0.3-0.5 (μm)×0.8-1.3 (μm). Itvigorously moves with one or more polar flagella.

B. Characteristics of Culture

1) Cultivation in meat infusion medium:

Growth of the bacteria was hardly observed. Off white translucentprecipitates formed very slightly at the bottom of the test tube.

2) Meat infusion agar stab culture:

Growth in thread form or small nipple form along the stab line wasobserved. Neither evolution of gas nor production of pigment wasobserved. Reddish thin bacterial plaque appeared on the surface, butthis bacterial plaque became translucent and light brown with the lapseof time and verrucose projections were observed in several places. It isan aerobic bacterium.

3) Meat infusion agar slant culture:

The growth of the bacteria was not so rapid and began at 28° C. aftertwo days (observed with naked eyes). The bacteria grew in thread form,and its bacterial plaque was translucent and light yellow with flatswelling having spotty appearance. The periphery was whole peripheral.Then, the bacterial plaque grew favorably in thread form or verrucoseform with gloss. Thus, a wet slightly reddished translucent brownbacterial plaque was obtained. The periphery was wavy or long wavy.Production of any gas or pigment was not observed.

4) Meat infusion gelatin stab culture:

Cultivation was effected at room temperature (22°-25° C.). The gelatinwas slightly liquefied.

5) Cultivation on the meat infusion agar plane medium:

The growth of the bacteria was not so rapid. The colony became visibleat 28° C. after two days. The colony was initially small, spotty,translucent and brown with a whole periphery. The colony was two smallto be observed about its swelling. Then, the colony grew in a spotty orcircular form and with whole periphery and the swelling was flat orconvex circular. The colony was translucent and brown with gloss.Neither gas nor soluble pigment was produced.

6) Characteristics in litmus milk culture:

An acid formation did not occur, and peptonization occurred but thereaction began after 14 days. Thus, the reaction was rather slowly. Nogas evolved.

C. Physiological and Biochemical Properties

1) Catalase test: positive

2) Oxidase test: positive

3) OF-test: negative (showing to be alkaline)

4) Hemolytic test: positive (weakly)

5) Viability at 5° C.: negative

6) Production of H₂ S: negative

7) Reducing ability for nitrate: positive

8) Denitrification: negative (although no nitrogen gas was evolved, itseemed to reduce No₂)

9) Availability of citric acid: negative (Christensen medium and Simonsmedium)

10) Growth on NAC agar medium: negative (nonviable)

11) Production of indole: negative

12) Voges-Proskauer reaction (Voges-Proskauer test): negative

13) Methyl Red test: negative

14) Hydrolyzing ability for arginine: weakly positive

15) Decarboxylation ability for lysine: positive

16) Decarboxylation ability for ornithine: positive

17) Hydrolyzing ability for esculin: negative

18) DNase test: negative

19) Hydrolyzing ability for starch: negative

20) ONPG test (cultivated at 37° C.): negative

21) Acylamidase test: positive

22) Phosphatase test: positive

23) Hydrolyzing ability for chitin: negative

24) Productivity of levan from sucrose: positive

25) Productivity of acids and gas from sugars: Neither acids nor gaseswere produced from the following 13 sugars: glucose, fructose,galactose, mannose, xylose, arabinose, maltose, lactose, rhamnose,sucrose, cellobiose, trehalose and mannitol.

26) Accumulation of poly-β-hydroxybutyrate in the cell: negative

27) Availability of carbon sources: On the medium containing minerals,glucose and calcium 2-keto-gluconate can be used as a sole carbon sourcefor the formation of the cells. In this case, it seemed that specificvitamins for growth were not required. On the other hand,D-(+)-trehalose, DL-arginine, geraniol, β-alanine, L-valine and inositolwere not be utilized.

28) G+C mole % (HPLC method): 60.4% (A+T mole %=39.6%)

In view of the above test results, PBJ-5360-STR-1-21 is an aerobicGram-negative rod and moves actively in a liquid medium using one ormore polar flagella. It is positive for catalase and contains oxidase.It was negative for OF-test (showing to be alkaline). In view of theseobservations, it is apparent that the present bacterium belongs to GenusPseudomonas in Family Pseudomonadaceae.

When the inventors compared the above properties with those of thebacterial complexes which are incapable of accumulatingpoly-β-hydroxybutyrate (PHB) in their cells, which complexes aredescribed in Bergey's Manual of Systematic Bacteriology, Vol. 1 (1984)on Genus Pseudomonas, the present inventors failed to find any bacteriumhaving those properties consistent or analogous to the propertiesdescribed above. This bacterium appears a considerably unusual strain ofPseudomonas because it hydrolyzes arginine and decarboxylates lysine andornithine. The G+C mole % value of 60.4% indicates that the strainbelongs to a group having lower G+C value in Pseudomonas. Thus, in viewof the various properties mentioned above, the present bacterium hasbeen identified as Pseudomonas sp. PBJ-5360-STR-1-21. These propertieswere consistent with those of the parent strain PBJ-5360.

What is claimed is:
 1. Stalobacin selected from the group consisting ofstalobacins A, B, C, D, E, F and G having physico-chemical properties asshown below in Tables 1, 2 and 3:

                  TABLE 1                                                         ______________________________________                                        Physico-chemical Properties of Stalobacins A and B:                                      Stalobacin A                                                                             Stalobacin B                                            ______________________________________                                        m.p. (°C.) (as Na salt)                                                             240° C. (dec.)                                                                      240° C. (dec.)                               LSI-MS Maximal                                                                             1483         1364                                                Peak (m/z)                                                                    HRLSI-MS     1483.7347    1364.7165                                           (MH.sup.+) (m/z)                                                                           C.sub.61 H.sub.107 N.sub.14 O.sub.28                                                       C.sub.58 H.sub.102 N.sub.13 O.sub.24                Theoretical Value                                                                          1483.7372    1364.7154                                           IR(KBr) (cm.sup.-1)                                                                        3385,1748    3389,1748                                                        1653,1526    1653,1526                                           UV(H.sub.2 O)                                                                              Terminal     Terminal Absorption                                              Absorption                                                       (ε) at 210 nm                                                                      36,300       35,500                                              CD           [θ].sub.196 - 77020                                                                  [θ].sub.193 - 70720                                        [θ].sub.212 + 3159                                                                   [θ].sub.209 + 17630                                        [θ].sub.231 - 33480                                                                  [θ].sub.231 - 29070                                        [θ].sub.257 + 4028                                                                   [θ].sub.257 + 4905                            Retention Time (min.)                                                                      7.8          8.4                                                 in HPLC*                                                                      Amino Acid Analysis (molar ratio)                                             HyAsp.sup.1) HyAsp(1)     HyAsp(1)                                            Asp          Asp(1)       Asp(1)                                              Ser          Ser(2)       Ser(1)                                              HyIle.sup.2) HyIle(1)     HyIle(1)                                            Gly          Gly(1)       Gly(1)                                              Ala          Ala(1)       Ala(1)                                              Arg          --           Arg(1)                                                           DNP - derivative                                                                           DNP - derivative                                                 m.p. >230° C.                                                                       m.p. >230° C. (dec.)                                      (dec.)                                                                        HRLSI-MS     HRLSI-MS 1530.7169                                               1649.7378                                                                     C.sub.67 H.sub.109 N.sub.16 O32                                                            C.sub.64 H.sub.103 N.sub.15 O.sub.28                             Theoretical Value                                                                          Theoretical Value                                                1649.7386    1530.7169                                           ______________________________________                                         *Column: Develosil 5C.sub.18, 4.6φ × 250 mm;                        Mobile Phase: CH.sub.3 CN/2 mM H.sub.3 PO.sub.4 (containing 50 mMNa.sub.2     SO.sub.4) = 43/57;                                                            Flow Rate: 1 ml/min.;                                                         Chart Speed: 1 cm/min.                                                        .sup.1) Hydroxyaspartic acid                                                  .sup.2) Hydroxyisoleucine                                                

                  TABLE 2                                                         ______________________________________                                        Physico-chemical Properties of Stalobacins C and D:                                    Stalobacin C Stalobacin D                                            ______________________________________                                        LSI-MS Maximal                                                                           1396           1309                                                Peak (m/z)                                                                    HRLSI-MS   1396.7061      1309.6706                                           (MH.sup.+) (m/z)                                                                         C.sub.58 H.sub.102 N.sub.13 O.sub.26                                                         C.sub.55 H.sub.97 N.sub.12 O.sub.24                 Theoretical Value                                                                        1396.7053      1309.6732                                           IR(KBr) (cm.sup.-1)                                                                      3411,1744      3418,1745                                                      1652,1528      1646,1525                                           UV(H.sub.2 O)                                                                            Terminal Absorption                                                                          Terminal Absorption                                 Retention Time                                                                           8.8            10.0                                                (min.) in HPLC*                                                               Amino Acid Analysis (molar ratio)                                             HyAsp.sup.1)                                                                             HyAsp(1)       HyAsp(1)                                            Asp        Asp(1)         Asp(1)                                              Ser        Ser(1)         --                                                  HyIle.sup.2)                                                                             HyIle(1)       HyIle (1)                                           Gly        Gly(1)         Gly(1)                                              Ala        Ala(1)         Ala(1)                                              Arg        --             --                                                  ______________________________________                                         *Column: Develosil 5C.sub.18, 4.6φ × 250 mm;                        Mobile Phase: CH.sub.3 CN/2 mM H.sub.3 PO.sub.4 (containing 50 mMNa.sub.2     SO.sub.4) = 43/57;                                                            Flow Rate: 1 ml/min.;                                                         Chart Speed: 1 cm/min.                                                        .sup.1) Hydroxyaspartic acid                                                  .sup.2) Hydroxyisoleucine                                                

                  TABLE 3                                                         ______________________________________                                        Physico-chemical Properties of Stalobacins E, F and G                                Stalobacin E                                                                            Stalobacin F                                                                              Stalobacin G                                     ______________________________________                                        m.p. (°C.) (as                                                                  240° C. dec.                                                                       240° C. dec.                                                                       240° C. dec.                          Na salt)                                                                      LSI-MS   1378        1497        1485                                         Maximal                                                                       Peak (m/z)                                                                    HRLSI-MS 1378.7318   1497.7532   1485.7529                                    (MH.sup.+) (m/z)                                                                       C.sub.59 H.sub.104 N.sub.13 O.sub.24                                                      C.sub.62 H.sub.109 N.sub.14 O.sub.28                                                      C.sub.61 H.sub.109 N.sub.14 O.sub.28         Theoretical                                                                            1378.7311   1497.7529   1485.7529                                    Value                                                                         IR(KBr)  3418,1748   3369,1746   3369,1746                                    (cm.sup.-1)                                                                            1651,1526   1653,1527   1654,1528                                    (UV) (H.sub.2 O)                                                                       Terminal    Terminal    Terminal                                              Absorption  Absorption  Absorption                                   (ε) at 210 nm                                                                  39,500      36,480      37,500                                       CD       [θ].sub.194 - 53530                                                                 [θ].sub.196 - 80050                                                                 [θ].sub.196 - 58600                             [θ].sub.208 + 15380                                                                 [θ].sub.212 + 1022                                                                  [θ].sub.212 + 10060                             [θ].sub.231 - 26750                                                                 [θ].sub.231 - 34490                                                                 [θ].sub.231 - 49830                             [θ].sub.256 + 4512                                                                  [θ].sub.257 + 4788                                                                  [θ].sub.257 + 5621                     Retention                                                                              12.4        11.7        17.0                                         Time (min.)*                                                                  in HPLC                                                                       Amino Acid Analysis (Molar Ratio)                                             HyAsp.sup.1)                                                                           HyAsp(1)    HyAsp(1)    HyAsp(1)                                     Asp      Asp(1)      Asp(1)      Asp(1)                                       Ser      Ser(1)      Ser(2)      Ser(2)                                       HyIle.sup.2)                                                                           HyIle(1)    HyIle(1)    HyIle(1)                                     Gly      Gly(1)      Gly(1)      Gly(1)                                       Ala      Ala(1)      Ala(1)      Ala(1)                                       Arg      Arg(1)      --          --                                           ______________________________________                                         *Column: Develosil 5C.sub.18, 4.6φ × 250 mm;                        Mobile Phase: CH.sub.3 CN/2 mM H.sub.3 PO.sub.4 (containing 50 mMNa.sub.2     SO.sub.4) = 43/57;                                                            Flow Rate: 1 ml/min.;                                                         Chart Speed: 1 cm/min.                                                        .sup.1) Hydroxyaspartic acid                                                  .sup.2) Hydroxyisoleucine                                                


2. Antibiotic stalobacin selected from the group consisting ofstalobacins H and I, produced by cultivating Pseudomonas sp.PBJ-5360-STR-1-21 in a culture medium until substantial antibioticactivity is imparted to the culture medium, and isolating a stalobacinhaving the physico-chemical properties shown below:

    ______________________________________                                                   Stalobacin H                                                                              Stalobacin I                                           ______________________________________                                        m.p. (°C.) (as Na salt)                                                             235° C. (dec.)                                                                       240° C. (dec.)                              LSI-MS Maximal Peak                                                                        1396          1325                                               (m/z)                                                                         IR (KBr) (cm.sup.-1)                                                                       3374, 1747,   3387, 1747,                                                     1654, 1597, 1525                                                                            1651, 1596, 1527                                   UV (H.sub.2 O)                                                                             Terminal absorption                                                                         Terminal absorption                                CD (H.sub.2 O)                                                                             [θ].sub.194 -66980                                                      [θ].sub.212 +9851                                                                     [θ].sub.206 +11530                                        [θ].sub.232 -31520                                                                    [θ].sub.232 -28660                                        [θ].sub.257 -4288                                                                     [θ].sub.257 +4749                            Retention time (min.)                                                                      8.8           9.7                                                in HPLC*                                                                      Amino Acid Analysis                                                           (molar ratio)                                                                 HyAsp.sup.1) HyAsp (1)     HyAsp (1)                                          Asp          Asp (1)       Asp (1)                                            Ser          Ser (1)       Ser (1)                                            HyIle.sup.2) HyIle (1)     HyIle (1)                                          Gly          Gly (1)       Gly (1)                                            Ala          Ala (1)       --                                                 ______________________________________                                         *Column: Develosil 5C18, 4.6 i.d. × 250 mm                              Mobile phase: CH.sub.3 CN/2 mM H.sub.3 PO.sub.4 (containing 50 mM Na.sub.     SO.sub.4) = 43/57                                                             Flow rate: 1 ml/min.                                                          .sup.1) Hydroxyaspartic acid                                                  .sup.2) Hydroxyisoleucine.                                               